Plasmids are double stranded, circular, extrachromosomal DNA molecules (plasmids are defined as such herein). Plasmids are contained inside host cells. One common host cell is Escherichia coli (E. coli). Many other types of cells are known to carry plasmids. This includes other bacteria, yeast, and higher eukaryotic cells. Plasmids may be artificial (i.e., manmade), such as cloning vectors carrying foreign DNA inserts. Plasmids may also occur naturally, such as in mitochondria and chloroplasts.
Since the invention of cloning circa 1975, the preparation of plasmid has been a routine task in molecular biology. Plasmid preparation has become a highly crowded art. The crowded nature of the art is a reflection of the widespread importance of the procedure in molecular biology. Numerous articles and patents have been published in the past 25 years describing novel methods for preparing plasmid. The problem of plasmid preparation has attracted enormous commercial interest. Companies sell kits for plasmid preparation (Amersham, Qbiogene, Clonetech, Promega, Biorad, Qiagen, Sigma); proprietary resins for purifying plasmid (Qiagen, Amersham, Puresyn, Macherey-Nagel); and automated instruments for preparing plasmid (Qiagen, MacConnell, Autogen).
In the purification of plasmid from host cells, usually bacterial cells, the final plasmid preparation is usually a mixture of two main forms of plasmid: open circular and supercoiled. In the supercoiled form, the plasmid has a covalently closed circular form, and the plasmid is negatively supercoiled in the host cell by the action of host enzymes. In the open circular form, one strand of the DNA duplex is broken at one or more places. The single strand break(s) in an open circular plasmid results in a relaxed topology.
Open circular plasmid in a plasmid preparation can result from several causes. Open circular plasmid may exist in the host cells immediately prior to lysis. Some supercoiled plasmid in the host cells may unintentionally be converted to open circular plasmid in the preparation of a cleared lysate, due to the fragile nature of supercoiled plasmid. Additional plasmid purification procedures, such as organic solvent extraction (e.g. phenol, chloroform), precipitation, ultrafiltration, and chromatography, may unintentionally convert some supercoiled plasmid from the cleared lysate to open circular plasmid, due to the fragile nature of supercoiled plasmid.
Within the context of this invention, unless otherwise indicated or implied, open circular plasmid refers to the open circular plasmid which is commonly present in plasmid preparations after purifying plasmid contained in host cells, and does not refer to open circular plasmid which is purposefully synthesized by an in vitro method. Such purposeful in vitro synthetic methods may be enzymatic or nonenzymatic reactions. Non-limiting examples of purposeful in vitro synthesis of open circular plasmid include purposeful in vitro plasmid replication forming open circular daughter plasmids, open circular plasmid purposefully synthesized from single stranded circular DNA by in vitro enzymatic reactions or synthetic primer annealing, and open circular plasmid produced by purposeful conversion of supercoiled plasmid to open circular plasmid such as purposeful damage with free radicals.
For most plasmid applications, the active plasmid form is supercoiled. Open circular plasmid is often either inactive or poorly active. Plasmid for gene transfer (e.g. in vitro DNA transformation or in vivo DNA therapy) may require a high percentage of supercoiled plasmid and a low percentage of open circular plasmid contamination. Numerous methods have been described in the prior art to achieve this objective.
Le Brun et al. described a method for purifying supercooled plasmid from open circular plasmid using agarose gel electrophoresis (BioTechniques 6:836-838, 1988). Separation was based on differential migration in agarose gel. Supercoiled plasmid was recovered from the ethidium bromide stained gel. Hediger described a similar method using continuous elution (Anal. Biochem. 159:280-286, 1986).
Gorich et al. described a method for purifying supercoiled plasmid from open circular plasmid using polyacrylamide gel electrophoresis (Electrophoresis 19:1575-1576, 1998). Separation was based on differential migration in polyacrylamide gel. Supercoiled plasmid was recovered from the gel by electrophoretic elution.
Womble et al. described a method for purfying supercoiled plasmid using density gradient centrifugation (J. Bacteriol. 130:148-153, 1977). Plasmid was dissolved in a cesium chloride-ethidium bromide solution and centrifuged at high speed. Supercoiled plasmid was separated from open circular plasmid based on differential incorporation of ethidium bromide.
Best et al. described a method for purifying supercoiled plasmid using reverse phase chromatography (Anal. Biochem. 114:235-243, 1981). The chromatographic resin separated supercoiled from open circular forms. Many chromatographic methods have been described in the prior art for separating supercoiled plasmid from open circular plasmid. This includes reverse phase, anion exchange, size exclusion, membrane, and thiophilic chromatography. Several chromatographic resins are commercially available for separating supercoiled from open circular forms (Puresyn, Amersham, Prometic).
Hyman described a method for purifying supercoiled plasmid using selective exonuclease digestion (BioTechniques, 13:550-554, 1992). A cell lysate was incubated with a mixture of exonuclease I and exonuclease III. The exonucleases selectively degraded open circular plasmid and chromosomal DNA without degrading supercoiled plasmid, thereby purifying supercoiled plasmid.
Prior art methods for purifying supercoiled plasmid from open circular plasmid involve separation and removal of open circular plasmid from supercoiled plasmid, or selective degradation of the open circular plasmid. In the chromatographic, electrophoretic, and ultracentrifugation prior art methods for purifying supercoiled plasmid, the open circular plasmid is separated and removed. In the enzymatic prior art methods, open circular plasmid is selectively degraded by exonuclease. One disadvantage of prior art approaches is that the final yield of supercoiled plasmid is reduced because open circular plasmid is removed or degraded.
The invention overcomes the inherent disadvantage of prior art methods by using a fundamentally different operating principle, by converting open circular plasmid to supercoiled plasmid. This invention provides an improved method for plasmid preparation.